This is a proposal to investigate the genetic control of patterned growth during the morphogenesis of a complex, multicomponent structure, the mammalian skull vault. More broadly, this proposal focuses on how interactions between distinct migratory mesenchymal cell populations regulate patterned growth. During skull vault development, cells derived from the neural crest and mesoderm migrate into positions above the eye, between the brain and a layer of non-osteogenic head mesoderm. There, in response to unknown cues they coalesce into the frontal and parietal bone rudiments and expand apically, ultimately coming into apposition with their paired counterparts. Although these events have been documented in broad outline, little is known about the developmental mechanisms that underlie them. Key issues that remain unresolved include whether cell migration has a role in rudiment elongation, how the pattern of the bones is determined, and what processes control the initial specification and differentiation of the frontal and parietal bone anlagen. Interactions between neural crest and mesoderm have long been postulated to be important in these processes, but how or even whether these interactions contribute to skull patterning remains unclear. Here we propose to address these questions. We have three Specific Aims: In the first, we will investigate the properties, cellular dynamics, and fate of a newly-defined population of migratory osteogenic precursor cells that contribute to the frontal and parietal bones. We will continue to carry out diI labeling experiments in conjunction with exo utero development of injected embryos to assess the fate and developmental function of migratory osteogenic precursor cells. We will evaluate a potential mechanism of frontal and parietal bone growth: that growth occurs by elongation of a rudiment of osteogenic cells through a preexisting layer of non- osteogenic mesenchyme. We will test the hypothesis that MOP cells originate in a population of noggin- expressing cells adjacent to the developing frontal and parietal bone rudiments In the second Specific Aim We will test the hypothesis that interactions between neural crest and mesoderm are essential for the growth of the calvarial rudiments and the anterior-posterior and dorso-ventral patterning of the frontal and parietal bones. We will continue to test the idea that Msx1/2 are required in the neural crest for the apical expansion of the frontal bone rudiment, and that changes in MOP cell life history contribute to dorso-ventral patterning defects of Msx mutants. We will also examine the mechanism by which the relative activity Msx2 in the neural crest and mesoderm controls the shape of the frontal and parietal bones, the location of the coronal suture, and thus patterning along the a/p axis. In the final Specific Aim, we will examine an interaction between FoxC1 and Msx1/2 that has revealed a signaling network by which the cell layers adjacent to the brain control the development of the frontal and parietal bone progenitor populations. This will entail a further characterization of FoxC1 mutants, as well as tests for a genetic interaction between Msx1/2 and FoxC1. We will also test of the hypothesis that FoxC1 interacts directly with a Bmp-responsive element in the Msx2 promoter, negatively regulating the response of Msx2 to Bmp signaling. The significance of the proposed work is that it addresses fundamental mechanisms of pattern formation as well as the pathophysiology of disorders affecting the growth and patterning of calvarial bones, including familial parietal foramina and craniosynostosis. PUBLIC HEALTH RELEVANCE: This is a proposal to study how the skull forms and how two genes-Msx2 and Msx2-control the shape of bones of the skull. Msx genes are of special interest because humans with mutations in MSX2 and MSX2 have birth defects that affect their skulls and teeth. By studying how these genes work, we will learn more about basic processes of skull development as well as how mutations in Msx1 and 2 lead to birth defects. In the long term, this work may help to devise new treatments of such birth defects.